O metabolismo da glutamina (Gln) é alvo de atenções recentes para a compreensão da reprogramação metabólica para o suprimento energético das células tumorais em proliferação e para o desenvolvimento de novas estratégias terapêuticas em câncer. Tanto a absorção de glutamina quanto a taxa de glutaminólise, o catabolismo da Gln para gerar adenosina trifosfato (ATP) e lactato na mitocôndria estão aumentados em diferentes tumores. A Gln e glicose participam do processo da proliferação de células tumorais tanto na produção de (ATP) como no fornecimento de produtos intermediários utilizados na síntese de macromoléculas e Gln é utilizado para anaplerose do ciclo do ácido tricarboxílico. Nesse estudo, nosso objetivo foi analisar a expressão dos genes envolvidos na glutaminólise: ASCT2, LAT1, GLS, GLSISO1, GLSISO2, GLS2, GOT1, GOT2, GLUD1 e GPT2 em astrocitomas de diferentes graus de malignidade (AGI-AGIV), classificados de acordo com a Organização Mundial de Saúde (OMS), em relação às expressões em tecidos cerebrais não neoplásicos e correlacionar os níveis de expressão destes genes aos dados clínicos. PCR quantitativo em tempo real (qRT-PCR) foi realizado em 175 amostras, sendo 22 dentre estes de tecidos não neoplásicos. Observou-se tempo de sobrevida menor entre os pacientes com hiperexpressão de LAT1, na presença de hipoexpressão de ASCT2. A expressão de GLS foi comparativamente maior que a expressão de GLS2 entre os astrocitomas de diferentes graus de malignidade, corroborando descrições prévias de que GLS relaciona-se à proliferação tumoral e GLS2 à supressão do crescimento tumoral. Observou-se, adicionalmente, o aumento da associação das expressões destes genes conforme o aumento do grau de malignidade, culminando em GBM, onde estas correlações foram estatisticamente significativas. Apesar da demonstração da ativação gradativa desta via da glutaminólise com o aumento da malignidade, a hiperexpressão dos genes relacionados a esta via mostrou-se hiperexpressa em apenas um subgrupo de pacientes com GBM. Esta observação ressalta a heterogeneidade observada em GBM e a elegibilidade restrita deste subgrupo a eventuais estratégias terapêuticas que forem desenvolvidas com alvos nesta via / The metabolism of glutamine (Gln) is the target of recent attentions to understand the metabolic reprogramming of cancer cell for the energetic needs for cell proliferation, and to develop new cancer therapeutic strategies. Glutamine absortion and glutamine conversion to ATP and lactate in the mitochondria through glutaminolysis are both increased in different cancer types. Gln and glucose participate in metabolic pathways which provide ATP and intermediate substrats for synthesis of macromolecules, and Gln is used for anaplesoris of tricyclic acid cycle. The aims of the present study were to analyze the differential mRNA expressions of genes involved in the glutaminolysis pathways: ASCT2, LAT1, GLS, GLSISO1, GLSISO2, GLS2, GOT1, GOT2, GLUD1 e GPT2 in astrocytomas of different grades of malignancy (WHO grades I to IV) compared to non-neoplastic brain tissues, and to correlate these expression data to clinical outcome. Shorter overall survival time was observed among a subset of GBM patients presenting hyperexpression of LAT1 while ASCT2 was hypoexpressed. GLS expression was comparatively higher than GLS2 expression among astrocytomas of different grades of malignancy, which corrobates previous reports relating GLS to tumor proliferation and GLS2 to suppression of tumor growth. Additionally, increased gene expression correlation was observed in parallel to the increase of malignancy, and these associated expressions were significant among GBM. Although a stepwise increase of the glutaminolysis pathway was demonstrated with the increase of malignancy in astrocytomas, the hyperexpression of genes involved in this pathway were detected only in a subset of GBM patients. This finding confirm the heterogeneity observed among GBM, and highlights the fact that any therapeutic strategy aiming this pathway will be restricted to a subset of GBM patients

O PAWR (Prostate apoptosis response- 4) também conhecido como PAR-4 é um gene pró-apoptótico identificado em células de câncer de próstata quando expostas a estímulos apoptóticos. A expressão de PAR-4 pode aumentar a sensibilidade da célula a apoptose, incluindo células de câncer de mama. Ensaios de expressão gênica por transcriptoma foram realizados em nosso laboratório (dados ainda não publicados), com o objetivo de analisar genes diferencialmente expressos associados à quimiossensibilidade ao docetaxel em células MCF-7 transfectadas com o vetor de expressão para PAR-4 (MCF7pcPAR4) e o com vetor vazio (MCF7pcNEO) antes e após o tratamento com docetaxel. Para avaliar a interação entre os genes diferencialmente expressos foram geradas redes gênicas funcionais utilizando o programa IPA- Ingenuity®. Dentre as diversas redes gênicas geradas destacaram-se as que continham genes relacionados direta ou indiretamente com a via de sinalização WNT (Wingless-Type MMTV Integration 1). O presente estudo visa validar genes diferencialmente expressos identificados em células MCF-7 com diferenças de expressão de PAR-4 antes e após a exposição de docetaxel. Através da anotação manual dos genes mais diferencialmente expressos, foram selecionados os genes EGR1, XAF1, TARP e WNT5A para validação por PCR em Tempo Real (qRT-PCR). A plataforma Human WNT Signaling Pathway RT2 Profiler(TM) PCR Array (PCR Array) foi utilizada para avaliar o efeito da overexpressão de PAR-4 e do tratamento de docetaxel na expressão de genes das vias canônica e não canônica do WNT. A expressão positiva do gene WNT5A nas células MCF7pcPAR4 em relação a MCF7pcNEO foi confirmada por qRT-PCR na ausença e presença do tratamento com docetaxel. O gene EGR1 apresentou regulação positiva significativa na técnica de qRT-PCR na ausência de tratamento, porém não apresenta o mesmo perfil de expressão observado no ensaio de transcriptoma. O gene XAF1 apresentou regulação negativa nas células MCF7pcPAR4 quando comparadas com as células MCF7pcNEO na ausência e presença de docetaxel, com tendência a validação na ausência de tratamento e de não validação na presença de docetaxel. Foi observado o aumento significativo da expressão de TARP nas células MCF7pcPAR4 por qRT-PCR na ausência e presença de docetaxel, porém esses achados não confirmam os resultados obtidos no transcriptoma. Em nossos dados de PCR Array na comparação MCF7pcPAR4 vs MCF7pcNEO antes do tratamento, encontramos diferença de expressão significativa (p < 0,005) em 9 genes. Sendo que, os genes CDKN2A, EGR1, FGF7, IL6 e TWIST apresentaram expressão positiva e os genes NTRK2, SOX2, SOX9 e WISP1 tiveram expressão negativa. Na comparação na presença de docetaxel observamos que os genes CACNAD2A3, GDF5, IL6, FGF7, LEF1 e TWIST apresentaram regulação positiva e FST apresentou regulação negativa com significância estatística (p < 0,005). Dos 84 genes da plataforma PCR array foram observados 21 e 14 genes comuns entre ambas às técnicas na ausência e presença de docetaxel, respectivamente. Na ausência de docetaxel 16 genes apresentaram o mesmo perfil de expressão, dentre estes a regulação positiva de GJA1, IGF1, IGF2, LEF1, MMP2, PDGFRA, PTGS2 e TWIST, associadas á regulação negativa de CDH1, JAG1, NTRK2 sugerem ativação da via WNT/?-catenina. Enquanto, a expressão de DAB2, WNT5A, FZD7 e RUNX2 indica inativação dessa via. Na presença do tratamento 6 genes apresentaram o mesmo perfil de expressão. Dentre estes, a regulação positiva de CTGF, DAB2 e EGR1 sugerem inativação da via WNT/beta-catenina. Por outro lado, a expressão positiva de FGF20, LEF1 e PDGFRA sugerem que via WNT/beta-catenina estaria ativa. Neste estudo, podemos mostrar que PAR-4 modula genes da via de sinalização WNT. Porém, mais experimentos serão necessários para verificar quais mecanismos estão envolvidos e de que forma isso reflete na quimiossensibilidade a drogas / PAWR (Prostate Apoptosis Response-4) also known as PAR-4 is a pro-apoptotic gene identified in prostate cancer cells when exposed to apoptotic stimuli. PAR-4 expression can increase sensitivity of cells to apoptosis, including breast cancer cells. Our laboratory performed transcriptome profiling (unpublished data), with the aim of analyzing differentially expressed genes associated with chemosensitivity to docetaxel in transfected MCF-7 cells with PAR-4 expression plasmid (MCF7pcPAR4) and with empty vector (MCF7pcNEO) before and after treatment with docetaxel. To assess the interaction between the differentially expressed genes functional gene networks were generated using the IPA Ingenuity® software. Networks generated containing genes directly or indirectly related with the WNT signaling pathway (Wingless-Type MMTV Integration 1) were highlighted. The present study aims to validate the differentially expressed genes identified in MCF-7 cells with different PAR-4 expression before and after exposure of docetaxel. By manual annotation of the genes most differentially expressed, the genes EGR1, XAF1, TARP and WNT5A were selected to be validated by quantitative real time PCR (qRT-PCR). The platform WNT Signaling Pathway Human RT2 Profiler (TM) PCR Array (Array PCR) was used to evaluate the effect of PAR-4 overexpression and docetaxel treatment in gene expression of canonical and noncanonical WNT pathways. The positive expression of WNT5A in MCF7pcPAR4 cells relative to MCF7pcNEO was confirmed by qRT-PCR in the presence and absence of docetaxel. The EGR1 gene showed significant upregulation in the qRT-PCR in the absence of treatment, but showed a different expression profile of the one observed in the transcriptome assay. The XAF1 showed a negative regulation in MCF7pcPAR4 cells when compared with MCF7pcNEO cells in the absence and presence of docetaxel, showing a similar trend in the absence of treatment but opposed trend in the presence of docetaxel. It was observed a significant increase in TARP expression in MCF7pcPAR4 cells by qRT-PCR in the absence and presence of docetaxel, but these findings do not confirm the results of the transcriptome. PCR Array data of MCF7pcPAR4 vs MCF7pcNEO comparison before treatment, showed a significant expression difference in nine genes (p < 0.005). The genes CDKN2A, EGR1, FGF7, IL6 and TWIST showed positive expression and the genes NTRK2, SOX2, SOX9 and WISP1 had negative expression. In the presence of docetaxel it was observed that the genes CACNAD2A3, GDF5, IL6, FGF7, LEF1 and TWIST showed upregulation and FST downregulation with statistical significance (p < 0.005). From 84 genes of the platform PCR array we observed 21 and 14 common genes between both techniques in the absence and presence docetaxel, respectively. In the absence of docetaxel 16 genes showed similar expression profile, among them the upregulation of GJA1, IGF1, IGF2, LEF1, MMP2, PDGFRA, PTGS2 and TWIST, together with downregulation of CDH1, JAG1, NTRK2 suggest activation of the WNT/beta-catenin pathway. While the expression of DAB2, WNT5A, FZD7 and RUNX2 indicates inactivation of this pathway. In the presence of treatment six genes showed the same expression profile. Among these, the upregulation of CTGF, DAB2 EGR1 suggest the inactivation of Wnt/beta-catenin pathway. On the other hand, positive expression of FGF20, LEF1 and PDGFRA suggests that Wnt/beta-catenin would be active. In this study, we show that PAR-4 modulates genes of the WNT signaling pathway. However, more experiments are needed to clarify the role of WNT canonical and non-canonical pathways and how this reflects on drug chemosensitivity

Iodoform (PI) and calcium hydroxide- based pastes (PH) are used in pulpectomy of primary teeth, complementing the antimicrobial action of mechanical chemical preparation and promoting tissue repair. These pastes take up and stay for a period of time along the root canal by contacting the periapical region and surrounding tissue, making it the aim of this study to quantify the relative expression of toxicity genes expressed by pastes, evaluating the biocompatibility these materials. By RT-PCR assay, were analyzed cellular mechanisms induced or generated by extracts of endodontic pastes: PI with Nebacetin (PNEB); with Maxitrol (PMAX); chlorhexidine (PCHX); and Guedes Pinto (GP); and PH: Calen® thickened with zinc oxide ( Calen®/ZO); and calcium hydroxide paste with Chlorhexidine (PHCHX); Ultracal®XS, using mononuclear cells from human peripheral blood (PBMC) , for to quantify the gene expression on the cytotoxic mediators: Granzyme A, Granzyme B, Granulysin and Perforin. Prior to gene expression, to analyze aspects of cell viability assays were performed - MTT, LDH and Capases Activity. The results were analyzed by Kolmogorov-Smirnov, one-way ANOVA and Tukey test, with p<0.05. The PNEB was the only one among all evaluated pastes that expressed all cytotoxic mediators above mentioned (p<0.05). Reviewed in cell viability, increased by PNEB Caspases 1 and 8 (p<0.05); The PGP increased Caspases 1, 3 and 8 (p<0.05); The PCHX decreased cell viability MTT and LDH, and exhibit increased activity of all Caspases (p<0.05). In PH, Ultracal®XS only presented significant concentration values for Caspase 1 , 3 and 8 (p<0.05). Despite the PNEB have presented high relative gene expression of all cytotoxic mediators analyzed, not caused decrease in cell viability, not causing increased activity of Caspase 3 (effector of apoptosis), demonstrating biocompatibility. Among the PI that showed better biocompatibility under the aspect of cytotoxicity were the PGP and PMAX. The material with cytotoxic potential observed in this experiment was the PCHX as dramatically decreased cell viability, confirmed the two tests and supported by high activity of all the analyzed Caspases, and also due to cell death, the inability of gene expression. All pH played citocompatível behavior, according to tests performed in the light of molecular biology. / Pastas iodoformadas (PI) e a base de hidróxido de cálcio (PH) são empregadas na pulpectomia de dentes decíduos, complementando a ação antimicrobiana do preparo químico mecânico e favorecendo o reparo tecidual. Essas pastas ocupam e permanecem por um período de tempo ao longo do canal radicular, entrando em contato com a região periapical e tecidos adjacentes, tornando-se o objetivo deste estudo quantificar a expressão relativa de genes de toxicidade manifestados pelas pastas, de forma a avaliar a biocompatibilidade das mesmas. Pelo ensaio RT-PCR, foram analisados mecanismos celulares induzidos ou gerados por extratos das pastas endodônticas: PI com Nebacetin (PNEB); com Maxitrol (PMAX); com Clorexidina (PCHX); e Guedes-Pinto (PGP); e PH: Calen® espessada com Oxido de Zinco (Calen®/ZOE); e pasta de Hidróxido de Cálcio com Clorexidina (PHCHX); Ultracal®XS, frente às células mononucleares do sangue periférico humano (CMSP), de forma a quantificar a expressão gênica relativa dos mediadores citotóxicos: Granzima A, Granzima B, Granulisina e Perforina. Previamente à expressão gênica, para analisar aspectos da viabilidade celular, foram realizados ensaios MTT, LDH e atividade de Caspases. Após teste de normalidade Kolmogorov-Smirnov, os resultados foram tratados por ANOVA de uma via e Tukey, com p<0,05. A PNEB foi a única pasta que expressou todos os mediadores citotóxicos avaliados (p<0.05). Com relação à viabilidade celular, a PNEB apresentou aumento de Caspases 1 e 8 (p<0.05); A PGP levou ao aumento das Caspases 1, 3 e 8 (p<0.05); A PCHX diminuiu a viabilidade celular no MTT e LDH, além de apresentar aumento da atividade de todas as Caspases (p<0.05). Nas PH, somente a Ultracal®XS apresentou valores de concentração significativos para Caspase 1, 3 e 8 (p<0.05). Apesar da PNEB ter apresentado elevada expressão gênica relativa de todos os mediadores citotóxicos analisados, não causou diminuição da viabilidade celular, não ocasionando aumento da atividade da Caspase 3 (efetora da apoptose), demonstrando biocompatibilidade. Dentre as PI que apresentaram melhor biocompatibilidade sob o aspecto de citotoxicidade foram a PGP e PMAX. O material com potencial citotóxico verificado neste experimento foi a PCHX, pois diminuiu drasticamente a viabilidade celular, confirmada em dois ensaios e corroborada pela alta atividade de todas as Caspases analisadas, e ainda, devido à morte celular, a incapacidade de expressão gênica. Todas as PH desempenharam comportamento citocompatível, de acordo com os ensaios realizados e à luz da biologia molecular.

The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The natural environments of organisms present a multitude of biotic and abiotic challenges that require both short-term ecological and long-term evolutionary responses. Though most environmental response studies have focused on effects at the ecosystem, community and organismal levels, the ultimate controls of these responses are located in the genome of the organism. Soil nematodes are highly responsive to, and display a wide variety of responses to changing environmental conditions, making them ideal models for the study of organismal interactions with their environment. In an attempt to examine responses to environmental stress (desiccation and freezing), genomic level analyses of gene expression during anhydrobiosis of the Antarctic nematode Plectus murrayi was undertaken. An EST library representative of the desiccation induced transcripts was established and the transcripts differentially expressed during desiccation stress were identified. The expressed genome of P. murrayi showed that desiccation survival in nematodes involves differential expression of a suite of genes from diverse functional areas, and constitutive expression of a number of stress related genes. My study also revealed that exposure to slow desiccation and freezing plays an important role in the transcription of stress related genes, improves desiccation and freezing survival of nematodes. Deterioration of traits essential for biological control has been recognized in diverse biological control agents including insect pathogenic nematodes. I studied the genetic mechanisms behind such deterioration using expression profiling. My results showed that trait deterioration of insect pathogenic nematode induces substantial overall changes in the nematode transcriptome and exhibits a general pattern of metabolic shift causing massive changes in metabolic and other processes. Finally, through field observations and molecular laboratory experiments the validity of the growth rate hypothesis in natural populations of Antarctic nematodes was tested. My results indicated that elemental stoichiometry influences evolutionary adaptations in gene expression and genome evolution. My study, in addition to providing immediate insight into the mechanisms by which multicellular animals respond to their environment, is transformative in its potential to inform other fundamental ecological and evolutionary questions, such as the evolution of life-history patterns and the relationship between community structure and ecological function in ecosystems.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

<p>Увод: Бројне генске мутације су у основи наследних склоности ка повећаном згрушавању крви (наследних тромбофилија). Најчешће испитиване наследне тромбофилије су: недостатак антитромбина, недостатак протеина Ц, недостатак протеина С, мутација проакцелерина FV Leiden и мутација протромбина FII G 20210 А. Тромбоза дубоких вена (ТДВ) често се јавља у општој популацији а код великог броја оболелих долази до поновне појаве болести. Стога је важно утврђивање значајности параметара који доприносе ризику за настанак ТДВ. Циљ: Утврђивање учесталости тромбофилних мутација код болесника са венским тромбоемболизмом и код здравих особа у популацији Војводине, испитивање повезаности тромбофилних мутација са локализацијом тромбозе и појавом плућне емболије, утврђивање разлике у времену појављивања ТДВ између оболелих са присуством појединачне и удружене тромбофилије, утврђивање учесталости удружене тромбофилије код оболелих и здравих особа и њен утицај на понављање тромбозног процеса у односу на појединачне тромбофилије и на основу добијених резултата утврђивање у којим случајевима је потребно извршити генетичко испитивање тромбофилије ради одређивања врсте и трајања антитромбозне терапије. Материјал и методе: Испитано је 175 оболелих особа са потврђеном ТДВ и 115 клинички и лабораторијски здравих особа које никад нису доживеле тромбозни инцидент и које су одабране тако да се контролна група поклапа са групом оболелих по полу. Активност антитромбина, протеина Ц и С, резистенција на активирани протеин Ц, ниво фактора VIII и ниво фибриногена одређивани су коагулационим тестовима а присуство мутација FV Leiden, FII G 20210 А, MTHFRС 677 Т и MTHFRА 1298 С испитивано је методом алелске дискриминације на Real-Time PCR инструменту. Оболелима је мерен и крвни притисак и одређиван липидни статус у случајевима сумње на поремећај нивоа липида у крви. Добијени подаци су нумерички обрађивани стандардним процедурама дескриптивне статистике и статистичке анализе. Резултати: Постојање наследне тромбофилије је утврђено код 57,14% оболелих са ТДВ и 48,21% здравих особа. Код укупно 16,57% оболелих испитаника утврђен је недостатак барем једног од природних инхибитора коагулације: код 4,0% недостатак антитромбина, код 1,71% недостатак протеина Ц, код 10,29% недостатак протеина С и код 0,57% истовремено постојање недостатка протеина Ц и протеина С. Постојање резистенције на активирани протеин Ц утврђено је код 30,38% оболелих. Постојање FV Leiden мутације утврђено је код 21,14% оболелих, код 16,0% у хетерозиготном а код 5,14% у хомозиготном облику и код 10,42% испитаника контролне групе у хетерозиготном облику док хомозиготних носилаца није било. Постојање FIIG 20210 А мутације утврђено је код 7,43% оболелих, код 4,0% у хетерозиготном облику а код 3,43% у хомозиготном облику и код 8,18% испитаника контролне групе, код 6,36% у хетерозиготном облику а код 1,82% у хомозиготном облику. Постојање мутације MTHFR С 677 Т је утврђено код 50,86% оболелих, код 42,86% у хетерозиготном облику а код 18,0% у хомозиготном облику и код 62,62% испитаника контролне групе, код 43,93% у хетерозиготном облику а код 18,69% у хомозиготном облику. Постојање мутације MTHFR А 1298 С утврђено је код 44,94% оболелих и 25,37% здравих испитаника са MTHFR С 677 Т мутацијом. Присуство фактора VIII у повишеној концентрацији утврђено је код 17,71% оболелих а присуство повишених вредности концентрац ије фибриногена код 9,81% оболелих. Код укупно 84,57% оболелих испитаника утврђено је постојање барем једне тромбофилије. Закључци: Између постојања недостатака природних инхибитора коагулације, постојања FV Leiden мутације у хомозиготном облику, као и постојања удружене наследне тромбофилије и настанка ТДВ утврђено је постојање статистички значајне повезаности. Недостатак неког од природних инхибитора повезан је са највећим ризиком за настанак ТДВ. Између постојања FIIG 20210 А мутације, у хомозиготном или хетерозиготном облику, постојања FV Leiden мутације у хетерозиготном облику, као и постојања MTHFRC677 T мутације, самостално или истовремено са MTHFRА 1298 C мутацијом и настанка ТДВ није утврђено постојање статистички значајне повезаности. Између присуства FIIG 20210 А мутације или FV Leiden мутације и MTHFRC677 T мутације у хомозиготном облику, као и удружене наследне тромбофилије и појаве ТДВ у трудноћи или пуерперијуму утврђено је постојање статистички значајне повезаности. Између постојања наследне тромбофилије, појединачне или удружене, и клиничког испољавања венске тромбоемболијске болести код оболелих са ТДВ није утврђено постојање статистички значајне повезаности. Између постојања наследне тромбофилије, појединачне или удружене, и старости у време настанка ТДВ није утврђено постојање статистички значајне повезаности. Између присуства MTHFRC677 T мутације и поновљеног тромбозног процеса утврђено је постојање статистички значајне повезаности. Између присуства FIIG 20210 А мутације и FV Leiden мутације, у хомозиготном или хетерозиготном облику, удружене наследне тромбофилије и поновљеног тромбозног процеса није утврђено постојање статистички значајне повезаности. Између гојазности и повишеног крвног притиска и настанка ТДВ утврђено је постојање статистички значајне повезаности. Између хиперлипопротеинемије и настанка ТДВ није утврђено постојање статистички значајне повезаности. На основу добијених резултата можемо закључити да се одлука о испитивању наследне тромбофилије доноси узимајући у обзир старост, пол и присуство пролазних или трајних фактора ризика.</p> / <p>Uvod: Brojne genske mutacije su u osnovi naslednih sklonosti ka povećanom zgrušavanju krvi (naslednih trombofilija). Najčešće ispitivane nasledne trombofilije su: nedostatak antitrombina, nedostatak proteina C, nedostatak proteina S, mutacija proakcelerina FV Leiden i mutacija protrombina FII G 20210 A. Tromboza dubokih vena (TDV) često se javlja u opštoj populaciji a kod velikog broja obolelih dolazi do ponovne pojave bolesti. Stoga je važno utvrđivanje značajnosti parametara koji doprinose riziku za nastanak TDV. Cilj: Utvrđivanje učestalosti trombofilnih mutacija kod bolesnika sa venskim tromboembolizmom i kod zdravih osoba u populaciji Vojvodine, ispitivanje povezanosti trombofilnih mutacija sa lokalizacijom tromboze i pojavom plućne embolije, utvrđivanje razlike u vremenu pojavljivanja TDV između obolelih sa prisustvom pojedinačne i udružene trombofilije, utvrđivanje učestalosti udružene trombofilije kod obolelih i zdravih osoba i njen uticaj na ponavljanje tromboznog procesa u odnosu na pojedinačne trombofilije i na osnovu dobijenih rezultata utvrđivanje u kojim slučajevima je potrebno izvršiti genetičko ispitivanje trombofilije radi određivanja vrste i trajanja antitrombozne terapije. Materijal i metode: Ispitano je 175 obolelih osoba sa potvrđenom TDV i 115 klinički i laboratorijski zdravih osoba koje nikad nisu doživele trombozni incident i koje su odabrane tako da se kontrolna grupa poklapa sa grupom obolelih po polu. Aktivnost antitrombina, proteina C i S, rezistencija na aktivirani protein C, nivo faktora VIII i nivo fibrinogena određivani su koagulacionim testovima a prisustvo mutacija FV Leiden, FII G 20210 A, MTHFRS 677 T i MTHFRA 1298 S ispitivano je metodom alelske diskriminacije na Real-Time PCR instrumentu. Obolelima je meren i krvni pritisak i određivan lipidni status u slučajevima sumnje na poremećaj nivoa lipida u krvi. Dobijeni podaci su numerički obrađivani standardnim procedurama deskriptivne statistike i statističke analize. Rezultati: Postojanje nasledne trombofilije je utvrđeno kod 57,14% obolelih sa TDV i 48,21% zdravih osoba. Kod ukupno 16,57% obolelih ispitanika utvrđen je nedostatak barem jednog od prirodnih inhibitora koagulacije: kod 4,0% nedostatak antitrombina, kod 1,71% nedostatak proteina C, kod 10,29% nedostatak proteina S i kod 0,57% istovremeno postojanje nedostatka proteina C i proteina S. Postojanje rezistencije na aktivirani protein C utvrđeno je kod 30,38% obolelih. Postojanje FV Leiden mutacije utvrđeno je kod 21,14% obolelih, kod 16,0% u heterozigotnom a kod 5,14% u homozigotnom obliku i kod 10,42% ispitanika kontrolne grupe u heterozigotnom obliku dok homozigotnih nosilaca nije bilo. Postojanje FIIG 20210 A mutacije utvrđeno je kod 7,43% obolelih, kod 4,0% u heterozigotnom obliku a kod 3,43% u homozigotnom obliku i kod 8,18% ispitanika kontrolne grupe, kod 6,36% u heterozigotnom obliku a kod 1,82% u homozigotnom obliku. Postojanje mutacije MTHFR S 677 T je utvrđeno kod 50,86% obolelih, kod 42,86% u heterozigotnom obliku a kod 18,0% u homozigotnom obliku i kod 62,62% ispitanika kontrolne grupe, kod 43,93% u heterozigotnom obliku a kod 18,69% u homozigotnom obliku. Postojanje mutacije MTHFR A 1298 S utvrđeno je kod 44,94% obolelih i 25,37% zdravih ispitanika sa MTHFR S 677 T mutacijom. Prisustvo faktora VIII u povišenoj koncentraciji utvrđeno je kod 17,71% obolelih a prisustvo povišenih vrednosti koncentrac ije fibrinogena kod 9,81% obolelih. Kod ukupno 84,57% obolelih ispitanika utvrđeno je postojanje barem jedne trombofilije. Zaključci: Između postojanja nedostataka prirodnih inhibitora koagulacije, postojanja FV Leiden mutacije u homozigotnom obliku, kao i postojanja udružene nasledne trombofilije i nastanka TDV utvrđeno je postojanje statistički značajne povezanosti. Nedostatak nekog od prirodnih inhibitora povezan je sa najvećim rizikom za nastanak TDV. Između postojanja FIIG 20210 A mutacije, u homozigotnom ili heterozigotnom obliku, postojanja FV Leiden mutacije u heterozigotnom obliku, kao i postojanja MTHFRC677 T mutacije, samostalno ili istovremeno sa MTHFRA 1298 C mutacijom i nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Između prisustva FIIG 20210 A mutacije ili FV Leiden mutacije i MTHFRC677 T mutacije u homozigotnom obliku, kao i udružene nasledne trombofilije i pojave TDV u trudnoći ili puerperijumu utvrđeno je postojanje statistički značajne povezanosti. Između postojanja nasledne trombofilije, pojedinačne ili udružene, i kliničkog ispoljavanja venske tromboembolijske bolesti kod obolelih sa TDV nije utvrđeno postojanje statistički značajne povezanosti. Između postojanja nasledne trombofilije, pojedinačne ili udružene, i starosti u vreme nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Između prisustva MTHFRC677 T mutacije i ponovljenog tromboznog procesa utvrđeno je postojanje statistički značajne povezanosti. Između prisustva FIIG 20210 A mutacije i FV Leiden mutacije, u homozigotnom ili heterozigotnom obliku, udružene nasledne trombofilije i ponovljenog tromboznog procesa nije utvrđeno postojanje statistički značajne povezanosti. Između gojaznosti i povišenog krvnog pritiska i nastanka TDV utvrđeno je postojanje statistički značajne povezanosti. Između hiperlipoproteinemije i nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Na osnovu dobijenih rezultata možemo zaključiti da se odluka o ispitivanju nasledne trombofilije donosi uzimajući u obzir starost, pol i prisustvo prolaznih ili trajnih faktora rizika.</p> / <p>Introduction: Numerous mutations are involved in genetic basis of an increased tendency for blood to clot (inherited thrombophilia). Most commonly investigated are: antithrombin deficiency, protein C deficiency, protein S deficiency, proaccelerin gene mutation FV Leiden and prothrombin gene mutation FIIG 20210 А. Deep vein thrombosis (DVT) is common in the general population and many patients experience a recurrence of the disease. It is, therefore, important to determine the significance of parameters contributing to the increased risk for DVT development. Objective: The aim of this study was to determine frequencies of thrombophilic mutations in patients with venous thromboembolism and healthy subjects in population of Vojvodina, to evaluate the association between thrombophilic mutations and thrombosis localization including pulmonary embolism, to determine the difference in age of DVT occurrence between patients with single and combined thrombophilic abnormalities, to determine frequencies of combined thrombophilic abnormalities in patients and healthy subjects and their influence on DVT recurrence compared to single thrombophilic abnormalities and on the basis of the results to determine when genetic testing is needed in order to determine the type and duration of anticoagulation therapy. Materials and methods: The study included 175 patients with documented DVT and sex-matched group of 115 healthy subjects without clinical or laboratory abnormalities, never having experienced DVT. Antithrombin, protein C and protein S activity; activated protein C resistance, factor VIII and fibrinogen levels are determined using coagulation tests, while for FV Leiden, FII G 20210 А, MTHFR C 677 T and MTHFR A 1298C mutation detection allelic discrimination assays on Real-Time PCR instrument are performed. Blood pressure is measured on all patients and in case of suspected plasma lipid levels elevation lipid profile is determined. The data obtained were subject to descriptive statistic and statistical analysis. Results: The total incidence of inherited thrombophilia was 57,14% in patients and 48,21% in healthy subjects. Total frequency of natural coagulation inhibitor deficiencies in patients was 16,57%: antithrombin deficiency 4,0%, protein C deficiency 1,71%, protein S deficiency 10,29% and combined protein C and protein S deficiency 0,57%. The incidence of activated protein C resistance in patients was 30,38%. The incidence of FV Leiden mutation was 21,14% in patients, 16,0% were heterozygous and 5,14% were homozygous, and 10,42% in healthy subjects, all of which were heterozygous. The incidence of FIIG 20210 А mutation was 7,43% in patients, 4,0% were heterozygous and 3,43% were homozygous, and 8,18% in healthy subjects, 6,36% were heterozygous and 1,82% were homozygous. The incidence of MTHFR C 677 T mutation was 50,86% in patients, 42,86% were heterozygous and 18,0% were homozygous, and 62,62% in healthy subjects, 43,93% were heterozygous and 18,69% were homozygous. The incidence of MTHFR A 1298 C mutation was 44,94% in patients with MTHFR C 677 T mutation and 25,37% in healthy subjects MTHFR C 677 T mutation. At least one thrombophilic abnormalitiy was present in 84,57% patients. Conclusions: The association between natural coagulation inhibitor deficiencies, FV Leiden mutation in homozygous form and combined thrombophilia and DVT development was statistically significant. Natural coagulation inhibitor deficiencies were associated with greatest risk of DVT development. The association between FII G 20210 А mutation, in homozygous or heterozygous form, FV Leiden mutation in heterozygous form, MTHFR C 677 T mutation alone or combined with MTHFR A 1298 C mutation and DVT development was not statistically significant. The association between FII G 20210 А mutation, FV Leiden mutation and MTHFR C 677 T mutation in homozygous form and DVT development during pregnancy and puerperium was statistically significant. The association between single or combined inherited thrombophilia and thrombosis localization including pulmonary embolism was not statistically significant. The association between single or combined inherited thrombophilia and the age of DVT occurrence was not statistically significant. The association between MTHFR C 677 T mutation and DVT recurrence was statistically significant. The association between FII G 20210 А mutation and FV Leiden mutation, in homozygous or heterozygous form, combined thrombophilia and DVT recurrence was not statistically significant. The association between obesity and high blood pressure and DVT development was statistically significant. The association between hyperlipoproteinemia and DVT development was not statistically significant. Based on the results we can conclude that decision to perform inherited thrombophilia testing should be based upon age, gender and the presence of transient or permanent risk factors.</p>

Introdução: O linfoma difuso de grandes células B é o mais freqüente grupo de linfoma não- Hodgkin, perfazendo quase 50% dos casos no serviço de hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e Instituto do Câncer do Estado de São Paulo. Possui heterogeneidade clínica e biológica traduzida em mais de vinte subtipos na Organização Mundial da Saúde. Sua terapêutica se baseia na associação do anticorpo monoclonal anti-CD20 e quimioterapia com antracíclico, esquema que resulta em 43,5% de sobrevida global em 10 anos. Determinantes de prognóstico clínico como o Índice Internacional de Prognóstico e o Índice Internacional de Prognóstico Revisado carecem de acurácia, pois até 20% dos pacientes de baixo risco falecerão da doença e 60% dos pacientes de alto risco estarão vivos em quatro anos. Essas discrepâncias podem, em parte, ser atribuídas a fatores genéticos. A assinatura gênica do linfoma difuso de grandes células B tipo centro germinativo apresenta sobrevida global superior ao tipo células B ativadas (76% versus 16%, p=0,01), contudo o perfil de expressão gênica por microarray ainda não está disponível na prática clínica. Entretanto, o escore preditivo de mortalidade para linfoma difuso de grandes células B baseado no valor prognóstico da expressão dos genes BCL2, BCL6, CCND2, FN1, LMO2 e SCYA3 por PCR em tempo real quantitativa mostrou-se independente do Índice Internacional de Prognóstico na era pré-rituximabe. Mas não foi significante em pacientes de alto risco clínico tratados com R-CHOP. Os genes BCL2, CCND2 e SCYA3 integram a assinatura de células B ativadas, BCL6 e LMO2 a do centro germinativo e FN1 a linfonodal. Objetivo: Avaliar o impacto da expressão absoluta dos genes BCL2, BCL6, CCND2, FN1, LMO2 e SCYA3 em população brasileira com linfoma difuso de grandes células B tratada com R-CHOP em relação à resposta global, sobrevida livre de doença, sobrevida livre de progressão e sobrevida global. Métodos: A expressão gênica foi analisada por PCR em tempo real quantitativa de RNA extraído de amostras parafinadas de 63 pacientes, porém foi avaliável em 42. Seus valores foram normatizados pelo gene endógeno ABL e transformados em escala logarítmica na base 2 para posterior correlação com variáveis clínicas e de desfecho. Resultados: Com mediana de seguimento de 29 meses, as sobrevidas global, livre de doença e livre de progressão foram, respectivamente, 82,8%, 97,14% e 87,53%, enquanto a resposta completa foi 82,5%. A expressão de LMO2>3logs e BCL6>3,5logs definiu um grupo de maior sobrevida global (91% versus 64,3%, p=0,040) e sobrevida livre de doença (95,5% versus 70,7%, p=0,03), independentemente do Índice Internacional de Prognóstico (p=0,010 e p=0,042) e com significativa hiperexpressão do SCYA3 (p=0,046). Não se observou associação entre escore preditivo de mortalidade baseado nos seis genes e prognóstico. Assim, foi criado novo escore genético prognóstico baseado no poder da expressão concomitante de LMO2 e CCND2, definindo-se grupos de baixo risco (<2,5) e alto risco (>=2,5) com distintas sobrevidas global (92,4% versus 57,1%, p=0,011) e livre de progressão (96,2% versus 66,7%, p=0,013), independentes do IPI. Conclusão: Em pacientes com linfoma difuso de grandes células B tratados com R-CHOP, a hiperexpressão de BCL6, LMO2 e SCYA3 correlacionou-se com melhor prognóstico. O novo escore genético prognóstico definido por LMO2 e CCND2 estratificou grupos de risco de prognósticos distintos independentes do Índice Internacional de Prognóstico / Introduction: Diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma; which accounts for almost 50% of the cases at the Hematology Department of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and Instituto do Câncer do Estado de São Paulo. Its clinical and biological heterogeneity results in more than twenty subtypes according to the World Health Organization classification. Its treatment is based on a combination of anti-CD20 monoclonal antibody and antracycline-based chemotherapy, with a 10-year overall survival of 43.5%. Clinical prognostic determinants such as the International Prognostic Index and the Revised International Prognostic Index lack accuracy, since up to 20% of low-risk patients will die from the disease and up to 60% of high-risk patients will be alive within four years. Such discrepancies can partially be attributed to genetic factors. Diffuse large B-cell lymphoma germinal center gene signature shows superior overall survival compared to activated B-cell signature (76% versus 16%, p=0.01), however microarray gene expression profile is not yet available in clinical practice. Nonetheless, the Mortality Predictor Score for diffuse large B-cell lymphoma based on the prognostic value of BCL2, BCL6, CCND2, FN1, LMO2 and SCYA3 gene expression by quantitative real-time PCR has proved to be independent from the International Prognostic Index in the pre-rituximab era. But it was not significant in high clinical risk patients treated with R-CHOP. The genes BCL2, CCND2 and SCYA3 compose activated B-cell signature, whereas BCL6 and LMO2 compose the germinal center signature and FN1 the lymph-node signature. Objective: Evaluate the impact of BCL2, BCL6, CCND2, FN1, LMO2 and SCYA3 absolute gene expression in Brazilian population diagnosed with diffuse large B-cell lymphoma and treated with R-CHOP, with respect to overall response, disease free survival, progression free survival and overall survival. Methods: Gene expression was analyzed by quantitative real-time PCR of RNA extracted from paraffin-embedded samples of 63 patients, although evaluable in 42. Their values were normalized by endogenous gene ABL and log- transformed on a base 2 scale for subsequent correlation with clinical and outcome variables. Results: With a median follow-up of 29 months, overall survival, disease free survival and progression free survival accounted for 82.8%, 97.14% and 87.53% respectively, while complete response was 82.5%. The expression of LMO2>3logs and BCL6>3.5logs defined a group with higher overall survival (91% versus 64.3%, p=0.040) and progression free survival (95.5% versus 70.7%, p=0.03), independent of International Prognostic Index (p=0.010 and p=0.042) and with significant overexpression of SCYA3 (p=0.046). It was not identified any association between six gene Mortality Predictor Score and prognosis. As a result, we developed the New Genetic Prognostic Score based on the power of concomitant expression of LMO2 and CCND2, defining low-risk (<2.5) and high-risk (>=2.5) groups with distinct overall survival (92.4% versus 57.1%, p=0.011) and progression free survival (96.2% versus 66.7%, p=0.013), independent of International Prognostic Index. Conclusion: In patients with diffuse large B-cell lymphoma treated with R-CHOP, hyperexpression of BCL6, LMO2 and SCYA3 was correlated with a better prognosis. The New Genetic Prognostic Score, defined by LMO2 and CCND2, stratified risk groups with different prognosis, independent of International Prognostic Index

Introdução: A meningite tuberculosa (MTB) é a forma mais grave e fatal de tuberculose. O diagnóstico oportuno e o tratamento adequado e precoce são os principais fatores associados com o bom prognóstico. Os métodos utilizados na prática médica diária - achados clínicos, exames de imagem e análise de líquido cefalorraquidiano (LCR) - têm baixa acurácia. A pesquisa do DNA do Mycobacterium tuberculosis no LCR através da reação em cadeia da polimerase (PCR, do inglês polimerase chain reaction) com a metodologia nested é promissora, especialmente quando associada à praticidade da amplificação do DNA em tempo real. Objetivo: Avaliar o valor diagnóstico da nested PCR em tempo real (nRT-PCR, do inglês nested real-time PCR) na investigação de pacientes com MTB. Métodos: Estudo observacional realizado em duas fases: uma prospectiva e outra retrospectiva. Na fase prospectiva, foram incluídos pacientes com suspeita de MTB internados no Instituto de Infectologia Emílio Ribas (IIER). Informações clínicas, laboratoriais e radiológicas foram coletadas, assim como amostra de LCR de todos os pacientes. A partir de critérios internacionais padronizados, os pacientes foram categorizados como \"MTB Definitiva\", \"MTB Provável\", \"MTB Possível\" e \"Não MTB\". A nRT-PCR, utilizando o gene alvo mpt64, foi realizada em todas as amostras de LCR no Laboratório de Meningites Bacterianas do Instituto Adolfo Lutz. Sensibilidade, especificidade e intervalos de confiança (IC 95%) da nRT-PCR foram calculados com base no padrão-ouro (cultura positiva para M. tuberculosis ou isolamento de BAAR no sistema nervoso central) e nos pacientes com outros diagnósticos estabelecidos (Não MTB). Também foi calculada a proporção de pacientes com a nRT-PCR positiva em cada categoria clínica. Na fase retrospectiva, foi realizada uma revisão de prontuários de pacientes que tiveram a nRT-PCR solicitada no IIER e no Centro de Referência e Treinamento em DST/AIDS. Os mesmos procedimentos de categorização diagnóstica, cálculos de sensibilidade e especificidade foram adotados. Resultados: Na fase prospectiva, foram incluídos 102 pacientes, sendo 92 deles infectados por HIV. Nove deles tiveram o padrão-ouro positivo e foram classificados como \"MTB Definitiva\" e 81 deles tiveram outros diagnósticos estabelecidos (\"Não MTB\"). A sensibilidade e a especificidade da nRT-PCR foi 100% (IC95%:70-100 e 95-100, respectivamente). A positividade da nRT-PCR na categoria \"MTB Provável\" foi 50% (4/8 pacientes) e 25% na \"MTB Possível\" (1/4). Na fase retrospectiva, 56 pacientes foram incluídos, sendo 48 infectados por HIV. A nRT-PCR teve sensibilidade de 83% (5/6) e especificidade de 100% (0/45). A positividade na categoria \"MTB Provável\" foi 60% (3/5) e não houve pacientes classificados como \"MTB Possível\". Conclusão: A nRT-PCR apresentou boa sensibilidade e ótima especificidade, demonstrando seu valor diagnóstico na identificação oportuna de casos de MTB / Background: Tuberculous meningitis (TBM) is the most serious and lethal presentation of tuberculosis. Timely diagnosis and appropriated treatment are the main factors associated with good outcome. Methods used in the daily medical practice - clinical, radiological and cerebrospinal fluid (CSF) findings - have low accuracy. Search for Mycobacterium tuberculosis DNA in the CSF by polymerase chain reaction (PCR) using the nested methodology is promising, especially when combined with the practical approach of the real time DNA amplification. Objective: To evaluate the diagnostic value of a nested real-time PCR (nRT-PCR) in the investigation of patients with TBM. Methods: A two-phase observational study was carried out: prospective and retrospective. In the prospective phase, patients with suspected TBM hospitalized at \"Instituto de Infectologia Emílio Ribas\" (IIER) were included. Clinical, laboratory and radiological data were collected, as well as CSF samples of all patients. According to international standard criteria, patients were categorized as \"TBM Definite\", \"TBM Probable\", \"TBM Possible\" and \"Not TBM\". The nRT-PCR, using the mpt64 gene, was performed on all CSF sample in the Laboratory of Bacterial Meningitis, Adolfo Lutz Institute. Sensitivity, specificity and confidence intervals (95% CI) of the nRT-PCR were calculated based on the gold standard (culture positive for M. tuberculosis or AFB isolation on the central nervous system) and on patients with other established diagnoses (\"Not TBM\"). The proportion of patients with a positive nRT-PCR in each clinical category was also calculated. In the retrospective phase, medical chart review was performed in those patients who had the nRT-PCR requested in IIER and in the \"Centro de Referência e Treinamento em DST/AIDS\". The same diagnostic categorization and calculations of sensitivity and specificity were adopted. Results: 102 patients were included in the prospective phase, 92 of them HIV-infected. Nine of them had the gold standard positive and were classified as \"TBM Definite\" and 81 of them had other diagnoses established (\"Not TBM\"). The sensitivity and specificity of the nRT-PCR were 100% (95%CI: 70-100 and 95-100, respectively). The nRT-PCR positivity in category \"TBM Probable\" was 50% (4/8 patients) and 25% in \"TBM Possible\" (1/4). In retrospective phase, the nRT-PCR had a sensitivity of 83% (5/6) and specificity of 100% (0/45), among the 56 included patients (48 of them HIV infected). Positivity in \"TBM Probable\" category was 60% (3/5) and no patients were classified as \"TBM Possible\". Conclusion: The nRT-PCR showed good sensitivity and excellent specificity, showing its diagnostic value in the timely identification of TBM